Products of the Reaction of Phenyihydrazine With

Proc. NatL Acad. Sci. USA Vol. 78, No. 9, pp. 5508-5512, September 1981 Biochemistry

(3meso-Phenylbiliverdin IXa and N-phenylprotoporphyrin IX, products of the reaction of phenyihydrazine with oxyhemoproteins (neso-tolylbiliverdin/N-tolylprotoporphyrin/Heinz body anemia/oxyhemoglobin/oxymyoglobm) SETSUO SArrO AND HARVEY A. ITANO Department of Pathology, D-006, University of California, San Diego, La Jolla, California 92093 Contributed by Harvey A. Itano, June 12, 1981 ABSTRACT Oxyhemoglobin and oxymyoglobin were allowed EXPERIMENTAL PROCEDURES to react aerobically with phenylhydrazine and p-tolylhydrazine. The chloroform extract ofeach reaction mixture, after treatment Materials and Methods. To a solution of metmyoglobin (5 with H2SOdmethanol, yielded a blue pigment and a.green pig- g, type III from horse heart, Sigma) in potassium phosphate ment, which were identified by electronic absorption, mass, and buffer (pH 7.4, 0.01 M, 200 ml), a 50-fold excess of powdered proton NMR spectroscopy as the dimethyl esters of -mao-ar- Na2S204 was added under N2. This mixture was applied onto ylbiliverdin IXa and N-arylprotoporphyrin IX, respectively. N- a column of Sephadex G-25 (5 cm inside diameter x 30 cm) Phenyiprotoporphyrin IX dimethyl ester formed complexes, with equilibrated with phosphate buffer (pH 7.4, 0.01 M) and was Zn2+, Cd'+, and Hg'+ but not with other cations. The proton eluted from the column with the same buffer. The eluate was NMR spectrum of the zinc complex suggested binding of the dialyzed against phosphate buffer (pH 7.4, 0.1 M, 4 liters) to phenyl group to one ofthe two pyrrole rings ofprotoporphyrin IX obtain a solution ofoxymyoglobin (3.58 g, 72% yield). Washed with a propionic acid substitutent. The effectiveness ofphenylhy- human erythrocytes were lysed with 3 vol ofdistilled water and drazine as an inducer ofHeinz body formation may be due to de- centrifuged at 12,000 X g for 90 min to obtain a clear solution stabilization of the hemoglobin. molecule by the replacement of of hemolysate. This solution was dialyzed against phosphate heme with phenyl adducts ofbiliverdin and protoporphyrin. buffer (pH 7.4, 0.1 M, 4 liters), and the resulting dialysate was used for reactions of oxyhemoglobin. Biliverdin IX, obtained The green hemoglobin produced in the reaction ofhemoglobin by coupled oxidation ofhemin, myoglobin, or hemoglobin with with phenylhydrazine was found by Kiese and Seipelt (1) to ascorbic acid (7, 8), was esterified in 7% H2SO4 in methanol. * differ from verdoglobins produced by other reagents. Lemberg The isomers ofbiliverdin IX dimethyl ester were separated by and Legge (2) showed that biliverdin production was increased thin-layer chromatography (TLC) (9). Phenylhydrazine-HCl in the erythrocytes ofrabbits treated with phenylhydrazine, and and p-tolylhydrazine HCl (Aldrich) were recrystallized from they concluded that this in vivo result of phenylhydrazine ac- ethanol before use. SilicAR cc-7 Special (Mallinckrodt) was used tivity corresponded to the in vitro formation ofbiliverdin in the for column chromatography. Uniplate (Silica Gel G, Analtech) coupled oxidation ofhemoglobin and ascorbic acid. Beaven and was used for analytical and preparative TLC. Electron impact White (3), on the other hand, did not obtain biliverdin in the mass spectra were obtained on an LKB type 9000 spectrometer in vitro reaction of phenylhydrazine with oxyhemoglobin; in- at an ionizing energy of70 eV by the direct inlet method. Field stead they isolated a green pigment with an absorption band in desorption mass spectra were obtained on a Kratos/AEI MS- the Soret region suggestive of an intact porphyrin ring. 902 instrument. Optical spectra were recorded on a Cary model Studies from this laboratory indicated that substituents on 17 spectrophotometer. Proton nuclear magnetic resonance the benzene ring affected the hemolytic activity of substituted (NMR) spectra of samples in C2HC13 solution containing inter- phenylhydrazine and the binding ofsubstituted phenyldiazenes nal tetramethylsilane were recorded with a custom-designed to methemoglobin in parallel fashion (4). Subsequent studies 360-MHz spectrometer with 200-400 pulses in the Fourier- showed, however, that the hemolytic activity of ring-substi- transform mode. High-performance liquid chromatography was tuted nitrosobenzenes was not related to their affinities as li- carried out with the Altex model 334 system. A Whatman Par- gands ofdeoxyhemoglobin (5). tisil 10-PAC column (4.6 x 250 mm) preceded by a short Partisil A two-electron transfer to oxyhemoglobin was suggested as 10 protective precolumn was used. Mass spectra of the four the initial step in the degradation ofheme mediated by ascorbic isomers ofbiliverdin IX dimethyl ester and the NMR spectrum acid (6), and the same transfer was postulated in the reaction of biliverdin IXa dimethyl ester were obtained for use as ofphenylhydrazine with oxyhemoglobin (4). Although the pro- standards. cesses may be initiated in the same way, different reported Blue (I) and Green (II) Pigments from the Reaction ofPhen- products, biliverdin with ascorbic acid (2) and an intact por- ylhydrazine with Oxymyoglobin. A solution of phenylhydra- phyrin with phenyihydrazine (3), indicate that subsequent steps zine-HCl (188 mg) in phosphate buffer (pH 7.4, 0.1 M, 100 ml) are different. We therefore undertook to isolate and character- was added to 500 ml of260 jLM oxymyoglobin in the same buf- ize the products of heme degradation when hemoglobin is fer, and the reaction mixture was allowed to stand for 1 hr under treated with arylhydrazines, and we found two types of com- aerobic conditions with occasional shaking. Acetic acid (400 ml) pounds: one is a biliverdin IXa derivative with an aryl group and concentrated HC1 (100 ml) were then added to 0C. After on a meso carbon, and the other is a protoporphyrin IX deriv- 20 hr at 40C, the reaction mixture was extracted twice with chlo- ative with an aryl group on a pyrrole nitrogen. roform (300 ml and 150 ml). The extracts were gathered, washed The publication costs ofthis article were defrayed in part by page charge Abbreviations: TLC, thin-layer chromatography; Fe", deoxyhemopro- payment. This article must therefore be hereby marked "advertise- tein; FeII02, oxyhemoprotein. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * All reagent percentages and solvent ratios are vol/vol. 5508 Downloaded by guest on October 1, 2021 Biochemistry: Saito and Itano Proc. Natd Acad. Sci. USA 78 (1981) 5509 with distilled water, dried over anhydrous Na2SO4, filtered, and globin (227 pM) in phosphate buffer (pH 7.4, 0.1 M, 600 ml), evaporated to obtain residue A (99.5 mg, 59.9% yield from a solution ofphenylhydrazine-HCl (800 mg) in the same buffer phenylprotoporphyrin, Mr 638). A solution ofthis residue in 7% (100 ml) was added. The procedure described above to obtain H2S04 in methanol (100 ml) at 0C was allowed to stand for 18 residue A was used to obtain residue C (405 mg, 57.3% yield hr at 4TC, diluted with cold distilled water (150 ml), and ex- from phenylprotoporphyrin, Mr 638). The crude product from tracted twice with chloroform (200 ml and 100 ml). The extracts the treatment of residue C with 7% H2SO4 in methanol was were treated as described above to obtain a residue that showed applied onto a silica gel column (5 cm inside diameter X 20 cm) a minor blue spot (RF: 0.47 in 4:1 benzene/acetone, 0.90 in 22:3 and eluted with the following solvents: 100% chloroform (200 chloroform/ethanol), a major green spot (RF: 0.02 in 4:1 ben- ml), 5% acetone in chloroform (200 ml), 5% methanol in chlo- zene/acetone, 0.20 in 22:3 chloroform/ethanol), and some roform (200 ml), and 10% methanol in chloroform (400 ml). The brown spots by TLC. The residue was applied onto a silica gel 5% acetone eluate was evaporated to give a residue that showed column (1.5 cm inside diameter x 12 cm) and eluted in succes- a major blue spot (RF: 0.47 in 4:1 benzene/acetone and a minor sion with the following solvents: 100% chloroform (100 ml), 3% blue spot (RF: 0.53 in 4:1 benzene/acetone) that overlapped a acetone in chloroform (50 ml), 5% acetone in chloroform (50 ml), brown spot by TLC. The major blue pigment (43mg, 9.9% from 5% methanol in chloroform (150 ml), and 10% methanol in chlo- residue C) was isolated and purified by preparative TLC. This roform (300 ml). The 5% acetone eluate was evaporated to ob- blue pigment was the same as pigment I obtained from the re- tain a blue oil (I, 13 mg, 12% yield from residue A), which was action of oxymyoglobin with phenylhydrazine according to its further purified by preparative TLC (silica gel, 4:1 benzene/ TLC behavior, electronic absorption spectrum, and NMR spec- acetone) for spectral determinations. Mass spectrum m/e (rel- trum. The 10% methanol eluate was evaporated to give a res- ative intensity): 686 (parent ion M+, 2.0), 567 (14.0), 478 (3.6), idue from which a green pigment (184 mg, 43.5% yield from 404 (3.6), 402 (3.4), 390 (56.8), 376 (100), 361 (4.5), 359 (3.6), residue C) was isolated by preparative TLC. The TLC and spec- 347(4.0), 345 (5.9), 317 (20.5), 314 (9.8), 303 (20.0), 300 (36.0), tral properties of this green pigment were the same as those of 287 (50.0), 241 (22.7), 227 (40.9), 199 (27.3), 181 (22.6), 180 pigment II from the reaction of oxymyoglobin with (22.7), 122 (22.7), 155 (13.6), 108 (40.9). NMR spectrum (in phenylhydrazine. C2HC13; 6 in ppm; m, multiplet; s, singlet) 6: 1.60 (CH3), 1.94 Zinc Complex of Green Pigment II. Zinc acetate (500 mg) (CH3), 2.01 (CH3), 2.03 (CH3), 2.54 (4H, m, Ar-CH2 X 2), in methanol (20 ml) was added at room temperature to a chlo- 2.88 (4H, m, -CH2CO X 2), 3.68 (6H, OCH3 X 2), 5.77 (4H, roform solution (100 ml) of green pigment II (50 mg), and the m, -CH=CH2 X 2), 5.95 (1H, s, H-15), 6.60-6.70 (2H, m, solvents were removed by evaporation. The residue was ex- -CH=CH2 x 2), 6.67 (1H, s, H-10), 7.26 (5H, -C6H5), 8.50 tracted with chloroform, and the extract was evaporated to ob- (3H, broad s, NH X 3). Electronic absorption spectrum tain a residue that showed a major green spot (RF: 0.39) accom- AxCh nm(8mM): 373 (50.1), 631 (14.5). The 10% methanol panied by two small green spots (RF: 0.33 and 0.31) on TLC eluate was evaporated, and the resulting residue was purified (85:15 chloroform/methanol). The major component (33 mg, by preparative TLC (silica gel, 85:15 chloroform/methanol) to 60% yield) was isolated by preparative TLC and purified by obtain a green pigment (II, 42.3 mg, 40.7% yield from residue silica gel column chromatography. The zinc complex of green A). Field desorption mass spectrum m/e: 666 (M+), 667 (M+ pigment II eluted as a single peak by high-performance liquid + 1). NMR spectrum (in C2HC13) 8: 2.26 (2H ortho protons of chromatography (4:1:2 n-hexane/tetrahydrofuran/methanol). phenyl group), 2.80-3.30 (4H, -CH2CO X 2), 3.30-3.65 NMR spectrum (C2HC13;J coupling constant; dd, double doub- (18H, CH3 X 4 and OCH3 X 2), 4.20-4.50 (4H, Ar-CH2- let) 8: 2.00 (2H dd, J, = 14 Hz, 12 = 5 Hz, ortho protons of X 2), 4.75 (2H, meta protons ofphenyl group), 5.47 (1H, para phenyl group), 2.82 and 3.08 (4H --CH2CO- X 2), 3.42-3.63 proton of phenyl group), 6.10-6.65 (4H, -CH=CH2 x 2), (18H, OCH3 X 2 and aryl-CH3 x 4), 4.10-4.40 (4H, 7.80-8.25 (2H, -CH=CH2 X 2), 10.10-10.50 (4H, nwso pro- aryl--CH2- X 2), 5.01 (2H, dd, Ji = 14 Hz, 12 = 5 Hz, meta tons). Electronic absorption spectrum AmaxC nm (emM): free protons ofphenyl group), 5.57 (1H, dd, J, = 14 Hz, 12 = 5 Hz, base 426 (140.6), 517 (14.0), 550 (9.1), 612 (6.0), 670 (2.6); uni- para proton ofphenyl group), 6.08-6.45 (4H, m, -CH=CH2 valent cation 412 (166.3), 555(10.3), 574(15.8), 595(11.7), 615 X 2), 7.98-8.30 (2H, m, --CH=CH2 x 2), 10.05-10.25 (4H, (8.0); Zn2+ complex 442(158.3), 548(12.2), 602(14.2), 645(4.4); m, meso protons). Cd2+ complex 438(148.3), 565(12.0), 607(13.3), 653(3.8); Hg2+ complex 450 (143.3), 554 (12.4), 609 (14.0), 660 (4.7). Blue (I) and Green (IV) Pigments from the Reaction of p- RESULTS Tolyihydrazine with Oxymyoglobin. To a solution of oxymyo- Structure of Blue Pigments I and HI. Blue pigment I from globin (150,uM) in phosphate buffer (pH 7.4, 0.1 M, 300 ml), the reations of both oxymyoglobin and oxyhemoglobin with p-tolylhydrazine-HCl (117 mg) was added. The procedures de- phenylhydrazine and blue pigment MI-from the reaction ofoxy- scribed above for the phenylhydrazine reaction were followed myoglobin with p-tolylhydrazine gave electronic absorption to obtain residue B (54.7 mg, 55.9% yield from tolylprotopor- spectra similar to the spectrum of biliverdin IX dimethyl ester phyrin, Mr 652), a blue pigment (HI, 6 mg, 10.2% yield from (7, 10). The mass spectra ofI and III showed molecular ion peaks residue B), and a green pigment (IV, 32.8 mg, 57.4% yield from at m/e 686 and 700, respectively. These molecular ions sug- residue B). Blue pigment m mass spectrum m/e (relative in- gested that a phenyl group (mass unit 77) and a tolyl group (mass tensity): 700(M+, 3.1), 579 (6.5), 505(6.0), 404(40.0), 390 (100), unit 91) replaced a proton on biliverdin IX dimethyl ester (Mr 314 (7.0), 300 (20.0), 287 (4.0), 241 (3.1), 227 (6.0), 225 (4.2), = 610) in pigment I and pigment HI, respectively. The mass 213 (11.3), 181 (4.0). Green pigment IV field desorption mass spectra of biliverdin IXa dimethyl ester and biliverdin IXy di- spectrum m/e: 680 (M+) and 681 (M+ + 1). Electronic absorp- methyl ester show a characteristic fragment ion at m/e 300 (refs. tion spectrum FH3C1 nm (emM): free base 426(127.3), 518(13.3), 7 and 10 and present work), consistent with structure Va of Fig. 550(9.0), 612 (6.8), 670(2.8); univalent cation 420(159.4), 555 1. On the other hand, the mass spectra of biliverdin IX(3 di- (10.8), 573 (14.8), 595 (10.0), 523 (8.4); Zn2+ complex 432 methyl ester and biliverdin 1X6 dimethyl ester show a signifi- (141.0), 547 (9.9), 602 (14.0), 643 (4.5). cant ion at m/e 360 (ref. 7 and present work), consistent with Blue (I) and Green (II) Pigments from the Reaction ofPhen- structure VI. The mass spectra of pigments I and m showed ylhydrazine with Oxyhemoglobin. To a solution of oxyhemo- characteristic fragment ions as base peaks at m/e 376 and m/e Downloaded by guest on October 1, 2021 5510 Biochemistry: Saito and Itano Proc. Natl. Acad. Sci. VSA 78 (1981) Table 1. Comparison of chemical shifts from proton NMR spectra at 360 MHz 8, ppm (no. of hydrogens) 0 N H ] [ oH] Biliverdin IXa H R H H H Protons* ester Blue I Me dimethyl pigment P = -CH2CH2CO2CH3 Aryl methyl 1.85 (3H), 2.06 (3H), 1.60 (3H), 1.94 (3H), Va, R= H, m/e 300 VI, m/e 360 2.11 (3H), 2.16 (3H) 2.01 (3H), 2.03 (3H) Vb, R=4to ,m/e 376 Ester methyl 3.68 (6H) 3.68 (6H) meso H-5, H-15 5.96 (1H), 6.00 (1H) 5.95 (1H) Vc R==-3-CH3,m/e 390 meso H-10 6.76 (1H) 6.67 (1H) Vinyl 5.38 and 5.61 (2H) 5.48-5.67 (4H) FIG. 1. Characteristic fragment ions of biliverdin IX dimethyl es- -CH-CH2 5.98 and 6.06 (2H) ters and blue pigments I and III. Vinyl 6.50 (1H), 6.62 (1H) 6.36-6.50 (2H) -CH=CH2 .390, consistent with structures Vb and Vc, in which a phenyl * See Figs. 2 and 3 for structures and proton designations. group and a tolyl. group, respectively, are attached to ion Va. These data indicate that pigments I and mI are derivatives of three meso protons; the spectrum ofpigment I shows only two biliverdin dimethyl esters of type IXa or IXy, not of type IV3 meso protons, of which one is the y proton and the other is or IX8. either the (3 or the 8 proton. In biliverdin IXa, the 8 position The NMR spectrum ofblue pigment I, shown in Fig. 2, dif- is between two methyls, while the (3 position is between a fers significantly from that ofbiliverdin IXy dimethyl ester (11, methyl and a vinyl. In the spectrum of pigment I, the signals 12). While the spectrum is similar to that of biliverdin IXa, of one ofthe four methyls and one of.the two vinyls are shifted differences in signal patterns are discernible in the regions of upfield. These shifts indicate that the phenyl group is on the the protons on the meso carbons and pyrrole methyl groups. ,(nmeso carbon. Accordingly, pigment I is /3-meso-phenylbiliv- Differences between these NMR spectra are summarized in erdin IXa methyl ester, and pigment Im is 3-meso-p-tolylbiliv- Table 1. The spectrum ofbiliverdin IXa dimethyl ester shows erdin IXa dimethyl ester (Fig. 3).

COOCH3 .COOCK3 CH?2 CH2 H2 CH2 H3C CH H3C 6H2 CHE2 CH3 H3C CH 2,13,17-CH3

0 24-/7 \ 5 H 'C6H5H H H H | | |7-CH3

-CH: :CH2 , n5 NH

II I I l 9 8 7 6 5 4 3 2 1 0 8, ppm FIG. .2. Proton NMR spectrum at 360 MHz off3-meso-phenylbiliverdin IXa dimethyl ester in C2HC13. Downloaded by guest on October 1, 2021 Biochemistry: Saito and Itano Proc. NatL Acad. Sci. USA 78 (1981) 5511 at m/e 680 (M+) and 681 (M+ + 1). These results are consistent with the substitution ofone phenyl group (mass unit 77) and one tolyl group (mass unit 91), respectively, for a proton in protoporphyrin IX dimethyl ester (Mr 590). All ofthe carbon protons ofprotoporphyrin IX dimethyl ester (13) were assignable on the NMR spectrum ofthe zinc complex (Fig. 4). The protons ofthe ortho, reta, and para positions ofthe phenyl group, which are IMe IMe pjMe confirmed by the spin decoupling method, were shifted to rnme__~= -i.Hi2U"2IU.2p3ur ru higher fields at chemical shifts of 6 = 2.00, 5.01, and 5.57, I , R= I , R respectively. The signal patterns of the vinyl group at 6 6.09-6.45 and m, R= {3>CH3 IV, R =-0-CH3 7.98-8.30 are similar to those of protoporphyrin IX dimethyl ester (13); on the other hand, the signals ofthe methylene pro- FIG. 3. Structures of 8-meso-arylbiliverdins Ia dimethyl ester tons ofthe propionic acid methyl ester group are different. The (blue pigments I and I) and N-arylprotoporphyrin IX dimethyl ester (green pigments II and IV). signals of the methylene protons adjacent to the pyrrole ring (6 4.10-4.40) are broadened, and the signals of the methylene protons adjacent to the ester group are separated at chemical Structure of Green Pigments II and IV. Green pigment shifts of 2.82 and 3.08. These results suggest that the phenyl HIwas obtained from the reactions of both oxymyoglobin and group is attached to one ofthe two pyrrole rings with a propionic oxyhemoglobin with phenylhydrazine. Its field desorption mass acid methyl ester substituent (14, 15). spectrum showed peaks at m/e 666 (M') and 667 (M' + 1). The Metal ions (Mg2+, V4+, Cr3, Mn2, Fe2, Co2+, Cu2+, field desorption mass spectrum of green pigment IV from the Zn2+, Cd2+, Hg2+, Sn2 , Sb2+, A13+) were added to chloroform reaction of oxymyoglobin with p-tolylhydrazine showed peaks solutions ofII; only Zn2+, Cd2 , and Hg2e formed complexes. The electronic absorption spectra ofthe free base, the univalent cation, and the Zn2+ complex of green pigment II are shown in Fig. 5. These spectra are like those of other N-substituted A I porphyrin derivatives (14, 16, 17). Similar spectra were obtained with green pigment IV. Green pigments II and IV are therefore N-phenylprotoporphyrin IX dimethyl ester and Np- Il tolylprotoporphyrin IX dimethyl ester, respectively (Fig. 3). I DISCUSSION 11I The formation of f,-meso-phenylbiliverdin IXa requires two site-specific processes, phenylation of the 3-mneso carbon and oxidative cleavage ofthe a-meso position ofprotoporphyrin IX. Specific arylation ofthe /3-mwso carbon is a novel reaction, but specific a cleavage occurs both in the physiological degradation of heme and in the coupled oxidation of myoglobin and ascor- bate (8). A two-electron reduction of oxyhemoglobin (FeI02) was postulated as the initial reaction in coupled oxidation (18). The transient product ofthis reaction, formulated as Fe"H202, 60 8 6 4 2 0 is the immediate precursor of meso-hydroxyheme, the first 8, ppm chemically identifiable intermediate in the oxidative cleavage ofheme (6). This postulated mechanism was confirmed by the B I,ldemonstration ofneso oxidation when pyridine octaethylhem- I1

IE S

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 8, ppm Wavelength , nm FIG. 4. (A) Proton NMR spectrum at 360 MHz of zinc N-phenyl- FIG. 5. Electronic absorption spectra of N-phenylprotoporphyrin protoporphyrin IX dimethyl ester in C2HC13. (B) Decoupling of phenyl IX dimethyl ester: -, free base; ----, univalent cation; -.-, zinc protons and sidechain methylene protons. complex. Downloaded by guest on October 1, 2021 5512 Biochemistry: Saito and Itano Proc. Nad Acad. Sci. USA 78 (1981) ochrome was allowed to react with H202 (19). Fe"H202 has also acid residues that surround them (24). When an inherited amino been represented as its anhydro derivative, FeNvO (20). acid replacement alters these contacts, the molecule becomes The presence ofmeso-phenylbiliverdin coupled with the ab- unstable and precipitates as Heinz bodies, the formation of sence of unsubstituted biliverdin among the products of the which is associated with hemolytic anemia (25). Some small reaction between phenylhydrazine and myoglobin indicates a compounds induce Heinz body hemolytic anemia even with close linkage between oxidative cleavage and meso phenylation normal hemoglobin; of these, phenylhydrazine is the most ef- in this reaction. Abimolecular reaction between oxyhemoglobin fective. Disruption ofnormal stabilizingcontacts between heme and phenylhydrazine (4) would simultaneously initiate both ring and globin when heme is replaced with meso-phenylbiliverdin, cleavage and ring phenylation. N-phenylprotoporphyrin, or both, may be the physical basis for the effectiveness ofphenylhydrazine. Fe"02 + C6H5NHNH2 -) [Fe"H202] + C6H5N:NH [1] We thank Mr. Kenneth M. Straub of the Bio-organic, Biomedical Fe"IH202 is the postulated intermediate that leads to meso ox- Mass Spectrometry Resource (National Institutes of Health Grant RR idation in coupled oxidation (6, 19). Phenyldiazene (C6H5N:NH) 00719; A. L. Burlingame, Director), University ofCalifornia, Berkeley, reacts rapidly with oxygen to become phenyldiazenyl radical forfield desorption mass spectra, and Mr. Thomas S. Arrhenius and Dr. (21), which immediately decomposes to phenyl radical and N2 Martin Tientze for proton NMR spectra at 360 MHz. This work was (22). supported by National Institutes of Health Grant AM 14982. 1. Kiese, M. & Seipelt, L. (1943) Naunyn-Schniedebergs Arch. Exp. C6H5N:NH + 02 -_ C6H5N:N' + H + 02- [2] PathoL Pharmakot 200, 648-683. 2. Lemberg, R. & Legge, J. W. (1942) Aust. J. Exp. BioL Med. Sci. C6H5N:N- -- C6H5- + N2 [3] 20, 65-68. 3. Beaven, G. H. & White, J. C. (1954) Nature (London) 173, Eqs. 1 and 2 accountfor the effect on °2 tension ofaddingphen- 389-391. ylhydrazine to a solution ofoxyhemoglobin. A sharp drop in 02 4. Itano, H. A., Hirota, K. & Vedvick, T. S. (1977) Proc. NatL Acad. concentration followed by a spontaneous restoration of nearly Sci. USA 74, 2556-2560. three-fourths of the drop was recorded (23). These effects are 5. Hirota, K., Itano, H. A. & Vedvick, T. S. (1978) Biochem.J. 174, attributable to the reduction of 02 to superoxide radical (02) 693-697. of 6. Lemberg, R., Cortis-Jones, B. & Norrie, M. (1938) Biochem. J. by phenyldiazene followed by the recovery 02 from 02- 32, 171-186. Oxidation in situ of phenyldiazene from Eq. 1 by 02 entering 7. Bonnett, R. & McDonagh, A. F. (1973) J. Chem. Soc. Perkin the heme crevice would provide a phenyl radical in the vicinity Trans. 1 881-888. of the protoporphyrin group undergoing nmso oxidation. 8. O'Carra, P. & Colleran, E. (1969) FEBS Lett. 5, 295-298. The formation of N-phenylprotoporphyrin requires that the 9. O'Carra, P. & Colleran, E. (1970) J. Chromatogr. 50, 458-468. phenyl group be attached to a pyrrole nitrogen before the pro- 10. Bonnett, R. & McDonagh, A. F. (1970) J. Chem. Soc. D. irreversible. A mechanism 238-239. cess of oxidative cleavage becomes 11. Stoll, M. S. & Gray, C. H. (1977) Biochem.J. 163, 59-101. for its formation must at the same time account for its relatively 12. Rasmussen, R. D., Yokoyama, W. H., Blumenthal, S. G., Bergs- high yield with respect to that of(-meso-phenylbiliverdin. The trom, D. E. & Ruebner, B. H. (1980) AnaL Biochem. 101, 66-74. reaction ofunmodified oxyhemoprotein with phenyldiazene or 13. Janson, T. R. & Katz, J. J. (1972)J. Magn. Reson. 6, 209-220. phenyl radical from the autooxidation of phenylhydrazine is 14. Ortiz de Montellano, P. R., Beilan, H. S. & Kunze, K. L. (1981) unlikely to result in such a yield because autooxidation is only Proc. NatL Acad. Sci. USA 78, 1490-1494. with ox- 15. Ortiz de Montellano, P. R., Beilan, H. S., Kunze, K. L. & Mico, about 0.2% as fast as the reaction of phenylhydrazine B. A. (1981)J. BioL Chem. 256, 4395-4399. yhemoglobin (23). Phenyl radical from Eqs. 1-3 may add to a 16. Jackson, A. H. & Dearden, G. R. (1973) Ann. N.Y.Acad. Sci. 206, pyrrole nitrogen instead of to the -mnwso carbon and result in 151-176. displacement of the iron atom of heme, which is essential for 17. De Matteis, F., Gibbs, A. H., Jackson, A. H. & Weerasinghe, S. meso cleavage (19). In the presence of an excess of phenylhy- (1980) FEBS Lett. 19, 109-112. drazine, a second molecule of this compound may react with 18. Foulkes, E. C., Lemberg, R. & Purdom, P. (1951) Proc. R. Soc. to restore and London Ser. B. 138, 386-402. Fe"H202 (or FeWvO) Fe", aborting ring cleavage 19. Bonnett, R. & Dimsdale, M. J. (1972)J. Chem. Soc. Perkin Trans. at the same time producing another molecule ofphenyldiazene. 1 2540-2548. + + 20. Goldberg, B., Stern, A. & Peisach, J. (1976)J. BioL Chem. 251, Fe"H202 + C6H5NHNH2-- Fe"I 2H20 C6H5N:NH 3045-3051. 21. Huang, P. C. & Kosower, E. M. (1968) J. Am. Chem. Soc. 90, or FeNVO + C6H5NHNH2 -> Fe"i + H20 + C6H5N:NH. [4] 2367-2376. Formation of a second molecule of phenyldiazene agrees with 22. Kasukhin, L. F. & Ponomarchuk, M. P. (1974) Chem. Phys. 3, were 136-139. the finding (23) that two molecules of 02 per oxyheme 23. Vedvick, T. S. & Itano, H. A. (1981) Biochim. Biophys. Acta 672, rapidly consumed in the reaction of an excess of phenylhydra- 214-218. zine with oxyhemoglobin. 24. Perutz, M. F. (1969) Proc. R. Soc. London Ser. B. 173, 113-140. Stability ofthe hemoglobin molecule depends partly on con- 25. Carrell, R. W. & Lehmann, H. (1969) Semin. HematoL 6, tacts between its heme groups and the side chains ofthe amino 116-132. Downloaded by guest on October 1, 2021